Sensitizing agents for cancer therapy, methods of use and methods for the identification thereof

Field of the invention

This invention relates to the field of cancer therapy and specifically sensitizing agents for cancer therapy, including, but not limited to, radio and chemotherapy. Also described herein is the novel target UROD (uroporphyrinogen decarboxylase), the down-regulation or inhibition of which results in increased sensitivity to cancer therapies.

Background

Head and neck cancer (HNC) is the eighth most common cancer worldwide, with an estimated annual global incidence of approximately 650,000 cases and .about.90,000 deaths attributed to this disease per year [1]. HNC comprises a diverse group of tumor types arising from the upper aerodigestive tract, including the lip, nasal and oral cavities, sinuses, pharynx, larynx, and other sites in this anatomical region [2]. The vast majority of HNC diagnoses (>90%) are of squamous epithelial cell origin (oral cavity, pharynx, larynx), and are thus termed head and neck squamous cell carcinomas (HNSCC) [2]. Nasopharyngeal carcinoma (NPC) is a less common distinct HNC in that >90% of cases harbor latent Epstein-Barr virus [3]. At the time of diagnosis, .about.30-40% of HNC patients typically have localized disease, >50% have associated regional disease, and .about.10% harbor distant metastases. In addition to the anatomic and molecular heterogeneity of HNC, most patients present with locally advanced disease, and/or suffer from other co-morbidities, rendering HNC particularly challenging to treat. Despite the advances in therapeutic options over the recent few decades, treatment toxicities and overall clinical outcomes have remained disappointing [4]. For all sites and stages in the head and neck region, 5-year survival rates average .about.50% [5].

Radiation therapy (RT) remains the primary curative modality for HNC. Even the most effective RT regimens achieve local control rates of 45-55%, with disease-free survival rates of only 30-40% for patients with locally advanced head and neck squamous-cell carcinomas (HNSCC) [6]. Furthermore, standard RT administering the maximal tolerable dose, limited by the surrounding critical normal tissues, yet is still associated with significant morbidity. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to surrounding normal cells, is critical to improving the therapeutic ratio of RT. The benefits of chemotherapy or molecularly-targeted agents combined with RT for HNC is strongly supported through the results from randomized trials and meta-analyses [7, 8]. However, these results remain modest; meta-analyses have documented concurrent RT with chemotherapy to offer an absolute survival advantage of only 4.5% at 5 years [7]. The 5-year overall survival rate of HNSCC patients treated with both RT and Cetuximab is still only 45.6% [8], underscoring a continued need for further improvement.

Novel molecular therapies for HNC have been developed and evaluated, ranging from adenovirus-mediated gene therapy [9-11] to anti-sense oligonucleotide (ASO) approaches involving systemically delivered Bcl-2 ASO combined with local tumor RT [12]. More recently, a rapid, cell-based phenotype-driven high-throughput screen (HTS) was developed for the large-scale identification of novel HNC cytotoxics, preferably with radiosensitizing activities [13, 14].

Ionizing radiation (IR) induces a myriad of physico-chemical changes at the cellular and molecular level [15], most of which have not yet been clearly elucidated, suggesting the existence of many unidentified radiosensitizing targets.

Summary of invention

In accordance with one aspect, there is provided a method for sensitizing a subject with cancer to a cancer therapy comprising administering to the subject a sensitizing amount of an agent that downregulates or inhibits UROD.

Preferably, the cancer is a head and neck cancer and the cancer therapy is one of radiation therapy and chemotherapy.

In accordance with a further aspect, there is provided a method for sensitizing a subject with cancer to a cancer therapy comprising downregulating or inhibiting UROD in cancer cells of the subject.

In accordance with a further aspect, there is provided use of an agent that downregulates or inhibits UROD for sensitizing a subject to a cancer therapy.

In accordance with a further aspect, there is provided use of an agent that downregulates or inhibits UROD in the preparation of a medicament for sensitizing a subject to a cancer therapy.

In accordance with a further aspect, there is provided a compound for sensitizing a subject with cancer to a cancer therapy comprising a UROD inhibitor or UROD downregulator.

In accordance with a further aspect, there is provided a method for identifying an agent that sensitizes a subject with cancer to a cancer therapy comprising screening for a compound that downregulates or inhibits UROD.

In accordance with a further aspect, there is provided a method of prognosticating a survival outcome to a cancer therapy of a subject with cancer comprising: providing a sample comprising cancer cells from the subject; and determining the level of UROD expression and/or activity in the cancer cells; wherein a relatively low level of UROD expression and/or activity compared to a control is correlated with an improved clinical outcome in response to cancer therapy.

In accordance with a further aspect, there is provided a method of diagnosing a subject with cancer comprising: providing a sample from the subject; and assaying the level of UROD expression and/or activity in the sample; wherein a relatively high level of UROD expression and/or activity compared to a control is correlated with cancer.

In accordance with a further aspect, there is provided a kit for diagnosing a cancer in or prognosticating a survival outcome to a cancer therapy of a subject with the cancer, comprising an assay for UROD expression and/or activity along with instructions for use.

In accordance with a further aspect, there is provided a method for sensitizing a subject with cancer to a cancer therapy comprising elevating the intracellular iron in cancer cells of the subject.

In accordance with a further aspect, there is provided a method for sensitizing a subject with cancer to a cancer therapy comprising administering an agent that elevates intracellular iron.

In accordance with a further aspect, there is provided a use of an agent that elevates the intracellular iron in cancer cells for sensitizing a subject to a cancer therapy.

In accordance with a further aspect, there is provided a use of an agent that elevates the intracellular iron in cancer cells in the preparation of a medicament for sensitizing a subject to a cancer therapy.

In accordance with a further aspect, there is provided a compound for sensitizing a subject with cancer to a cancer therapy comprising an elevator of intracellular iron.

Brief description of the figures

Embodiments of the invention may best be understood by referring to the following description and accompanying drawings. In the drawings:

FIG. 1 shows the identification of UROD as a novel radiosensitizing target via a siRNA-based high-throughput screen. (A) Preliminary screen of the Human siGENOME Druggable (6080 genes) and Protein Kinase (800 genes) siRNA Libraries at 2 Gy in transfected FaDu (human hypopharyngeal squamous cell cancer) cells. (B) 67 target sequences with potential radiosensitizing effects (>50% reduction in surviving fraction at 2 Gy vs. 0 Gy) were identified. Targets that decreased the surviving fraction by >30% in the absence of IR were not considered (grey box). Known radiosensitizing targets (grey circles); UROD (black circle); scrambled siRNA control (black triangle). (C) Clonogenic survival curves of FaDu cells transfected with scrambled control siRNA (siCTRL) or UROD siRNA (siUROD) for 48 h, then irradiated (0-6 Gy). Colonies were counted 12 days post-IR. *p<0.05 and **p<0.01, siCTRL vs. siUROD for each IR dose. (D) As in (C), but FaDu cells were transfected with a range of siRNA concentrations (0-60 nM), combined with IR (0-6 Gy) for Chou-Talalay combination index analyses. (E) Relative UROD mRNA levels in FaDu cells transfected with siCTRL or siUROD for 24, 48, and 120 h, as measured by qRT-PCR. **p<0.01, siCTRL vs. siUROD. (F) UROD protein expression was detected by immunoblotting at 24-72 h post-transfection. (G) FaDu cells were co-transfected with siRNA (siCTRL or siUROD) and plasmid DNA (empty vector control, pVector or siRNA-resistant rescue plasmid, pUROD) for 48 h, and then irradiated (4 Gy). Apoptotic fractions were assessed by flow cytometry 72 h post-IR. **p<0.01, siCTRL-pVector vs. siUROD-pVector or siUROD-pUROD.+-.IR. Each datum represents the mean.+-.SEM from three independent experiments.

FIG. 2 shows that the radiosensitizing effect of UROD knockdown is independent of porphyrin accumulation. (A) Heme biosynthetic pathway. ALA, .delta.-aminolevulinic acid; CPOX, coproporphyrinogen oxidase; PPOX, protoporphyrinogen oxidase; Fe, iron. (B) Porphyrin synthesis in mock-, siCTRL-, or siUROD-transfected FaDu cells was artificially induced with ALA (500 .mu.M, 4 h) prior to porphyrin extraction at 24 h post-transfection. Porphyrin levels were quantified spectrofluorometrically and normalized to total cell number. Representative spectral scans (575-750 nm) are shown. **p<0.01, siUROD vs. siCTRL or untreated.+-.ALA. (C) Fluorescent microscopy images of transfected cells.+-.ALA (500 .mu.M, 1 h). Mitochondria and nuclei were stained with MitoTracker Green and Hoechst 33342, respectively. Intracellular porphyrin excited with a wavelength of .about.400 nm emits red fluorescence at a peak of .about.635 nm. Scale bar, 10 .mu.m. (D) ALA-treated (250-1000 .mu.M, 4 h) and siCTRL- or siUROD-transfected (48 h-transfection) FaDu cells were irradiated (4 Gy), then cell viability was assessed 96 h later via MTS assay. **p<0.01, siCTRL vs. siUROD.+-.IR; untreated vs. ALA.+-.IR. In all cases, each datum represents the mean.+-.SEM from three independent experiments.

FIG. 3 shows that UROD down-regulation promotes radiation-induced cytotoxicity. (A) Flow cytometric DNA content analyses of siCTRL- or siUROD-transfected FaDu cells at 12-72 h post-IR (4 Gy). Representative histograms with gates for cell cycle distributions are shown. *p<0.05 and **p<0.01, siCTRL vs. siUROD.+-.IR at each time point. (B) Flow cytometric analyses of cellular .gamma.-H2AX expression levels in transfected FaDu cells at 0-240 min post-IR (4 Gy). **p<0.01, siCTRL vs. siUROD at each time point. (C) Representative images of .gamma.-H2AX nuclear foci formation in siCTRL- and siUROD-transfected FaDu cells 30 min post-IR. Scale bar, 10 .mu.m. (D) Flow cytometric analyses of caspase 9, 8, and 3 activation in siCTRL or siUROD-transfected FaDu cells at 12-48 h post-IR (4 Gy). *p<0.05 and **p<0.01, siCTRL vs. siUROD.+-.IR at each time point. (E) .DELTA..PSI..sub.M depolarization was quantified by flow cytometry 48 h post-IR in transfected FaDu cells. **p<0.01, siCTRL vs. siUROD.+-.IR. Each datum represents the mean.+-.SEM from three independent experiments.

FIG. 4 shows that siUROD-mediated radiosensitization enhances cellular oxidative stress. (A) Intracellular superoxide anions in siCTRL- or siUROD-transfected FaDu cells at 3-72 h post-IR (4 Gy) were detected by flow cytometry with dihydroethidium (DE). *p<0.05 and **p<0.01, siCTRL vs. siUROD.+-.IR at each time point. (B) Overall ROS levels in transfected FaDu cells were measured with CM-H.sub.2DCFDA at 3-72 h post-IR (4 Gy). *p<0.05 and **p<0.01, siCTRL vs. siUROD.+-.IR at each time point. (C) Superoxide radical levels in two transfected normal head and neck epithelial cells (NOP, normal oropharyngeal; NOE, normal oral epithelial) 72 h post-IR (4 Gy). **p<0.01, normals vs. FaDu at 72 h post-IR. (D) Overall ROS levels in transfected NOP and NOE cells 72 h post-IR (4 Gy). *p<0.05 and **p<0.01, normals vs. FaDu at 72 h post-IR. (E) Cell viability of siCTRL or siUROD-transfected FaDu, NOP, and NOE cells at 96 h post-IR (2 Gy) via MTS assay. **p<0.01, siCTRL vs. siUROD.+-.IR. (F) FaDu cells were transfected with siCTRL or siUROD and irradiated under normoxia (21% O.sub.2) or hypoxia (0.2% O.sub.2). Apoptotic fractions were assessed by flow cytometry 72 h post-IR. *p<0.05 and **p<0.01, normoxic vs. hypoxic treatments. (G) Relative mRNA expression of a panel of genes involved in cellular oxidative stress responses in siCTRL- or siUROD-transfected FaDu cells 48 h post-IR. Relative fold changes represent average .DELTA.Ct values normalized to those of .beta.-actin, then compared to siCTRL-transfected cells. **p<0.01, siCTRL vs. siUROD.+-.IR. Each datum represents the mean.+-.SEM from three independent experiments.

FIG. 5 shows that UROD knockdown induces intracellular iron accumulation. (A) Ferrous (Fe.sup.2+) and ferric (Fe.sup.3+) iron staining of siCTRL or siUROD-transfected FaDu cells at 48 h post-IR (4 Gy). Scale bar, 50 .mu.m. (B) Quantification of intracellular Fe.sup.2+ and Fe.sup.3+ levels from (A). Deep-purple areas and total area of cultured cells were measured. The ratio (% area) was calculated by dividing the sum of deep-purple areas by the sum of the total area from sections. *p<0.05 and **p<0.01, siCTRL vs. siUROD.+-.IR. (C) FaDu cells transfected with siCTRL or siUROD for 24 h were treated with deferoxamine (DFO; 5 .mu.M), and then irradiated (4 Gy) 24 h later. Apoptotic fractions were assessed by flow cytometry 72 h post-IR. **p<0.01, -DFO vs. +DFO treatments. Each datum represents the mean.+-.SEM from at least two independent experiments.

FIG. 6 shows the in vivo efficacy of UROD knockdown plus irradiation in HNC models. (A) Mock, siCTRL, or siUROD-transfected FaDu cells were implanted into the left gastrocnemius muscle of SCID mice, followed immediately by local RT (4 Gy). Each treatment group comprised of 9 mice. ***p<0.001, siUROD vs. mock or siCTRL.+-.RT. (B) FaDu tumors were established in SCID mice; once TLDs reached .about.8 mm, mice were randomly assigned to siCTRL, siUROD, siCTRL-plus-RT, or siUROD-plus-RT. Mice were intraperitoneally-injected with 600 pmol of jetPEI-complexed siRNA thrice a week for up to 2 weeks (white arrows). Local tumor RT (4 Gy) was delivered on days 5 and 13 post IP-injections (grey arrows). Each treatment group comprised of .gtoreq.5 mice. ***p<0.001, siUROD vs. siCTRL+RT. (C) UROD knockdown was assessed in FaDu tumors 24 h after the last treatment as described in (B). Excised tumors were subjected to immunoblotting for UROD expression. Western blots were quantified and relative fold changes in UROD protein levels were determined by normalizing to corresponding GAPDH loading controls, then compared to siCTRL-treated tumors. (D) UROD knockdown in tumors (black arrows) was also verified by immunohistochemistry. (E) Minimal differences in the average mice body weights for each treatment group from (B) indicated that the systemic siUROD-plus-local RT regimen was well-tolerated. Each datum represents the mean.+-.SEM from at least two independent experiments.

FIG. 7 shows the clinical relevance of UROD in human cancers. (A) Cell viability assessment of siCTRL or siUROD-transfected cancer cells at 96 h post-IR (2 Gy) via MTS assay. Human HNC (FaDu, C666-1, UTSCC-8, UTSCC-42a), cervix (SiHa, ME-180), breast (T47D), lung (A549), and prostate (DU-145) cancer cell lines. **p<0.01, siCTRL vs. siUROD.+-.IR. (B) Relative UROD mRNA expression in UTSCC-42a cells transfected with UROD-expressing plasmid (pUROD) or empty vector control (pVector) for 48 h, determined via qRT-PCR. ***p<0.001, pVector vs. pUROD. (C) UTSCC-42a cells transfected with pUROD or pVector for 48 h were irradiated (2 Gy). Apoptotic fractions were assessed by flow cytometry 72 h post-IR. Representative histogram of cell cycle distribution is shown. ***p<0.001, pUROD vs. pVector+IR. (D) Total RNA was extracted from 38 HNSCC patient tumor biopsies and 5 normal laryngeal and tonsillar epithelial tissues, and assessed for relative levels of UROD mRNA expression. Fold change was determined by normalizing to .beta.-actin levels, and comparing to the average from normal tissues. Solid line, mean fold change. *p<0.05, tumor vs. normal tissues. (E) Kaplan-Meier plot of disease-free survival (DFS) for the HNSCC patients from (D); trichotomized based on interquartile range (low, medium, vs. high levels of UROD mRNA expression). DFS was defined as absence of relapse or death, calculated from the time of diagnosis. Median follow-up time was 6.9 years (range 2.3-10.8 yrs). (F) Cell viability assessment of irradiated (2 Gy) primary normal human fibroblasts (MRC5, GM05757) and untransformed fibroblasts from PCT patients (GM01482, GM00977, GM00961, GM01041) 96 h post-IR via MTS assay. *p<0.05, MRC5 vs. PCT fibroblasts. (G) siCTRL- or siUROD-transfected FaDu cells were treated with increasing doses of Cisplatin (0.01-0.25 .mu.M), 5-FU (1-2.5 .mu.M), or Paclitaxel (PTX) (0.1 .mu.M) for 24 h, then assessed for cell viability 96 h later. ***p<0.001 and *p<0.05, siCTRL.+-.drug vs. siUROD.+-.drug. Each datum represents the mean.+-.SEM from three independent experiments.

Detailed description

In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, a person skilled in the art would understand when the invention may be practiced without certain specific details. Some methods herein have been described as a series of steps and a person skilled in the art will also understand that the steps may be performed in any logical order unless the context dictates otherwise.

Head and neck cancer (HNC) is a challenging disease due to its heterogeneity and complexity, often resulting in poor survival rates. Radiation therapy (RT) remains the primary curative modality for HNC. Even the most effective RT regimens however, achieve local control rates of 45-55%, with disease-free survival rates of only 30-40%. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to the surrounding normal tissues, is critical to improving cure rates with RT.

A siRNA-based high-throughput screen (HTS) was developed for the large-scale identification of novel genes that will selectively sensitize HNC cells to radiation. The preliminary screen identified 188 target sequences with potential radiosensitizing effects; the validity of the screen was corroborated by the identification of known radiosensitizing targets (e.g. ATM, ATR, Aurora-A kinase). To confirm the initial HTS results, FaDu cells (human hypopharyngeal squamous cell cancer) were transfected with the 188 siRNAs.+-.RT, and those that were cytotoxic without RT were eliminated, leaving 67 potential `hits`. Targets reducing surviving fraction by >50% at 2 Gy relative to their un-irradiated counterparts were selected. Corroboration of siRNA-mediated mRNA and protein knockdown were assessed using qRT-PCR and Western blotting, respectively.

A key regulator of the heme biosynthetic pathway, uroporphyrinogen decarboxylase (UROD), was thus identified as a potent radiosensitizer. Increased heme biosynthesis has previously been reported in tumor tissues with up-regulation of several regulatory proteins, including UROD. The broad applicability of this radiosensitization was exhibited in other HNC cell lines (nasopharyngeal and laryngeal squamous cancers), as well as other cancer models (cervix, breast, lung, and prostate carcinomas); no radiosensitization was observed in normal oral cavity or oropharyngeal epithelial cells.

Functional validation studies and in vitro characterization of mechanisms for radiosensitization were examined. These studies suggest an effect mediated by tumor-selective enhancement of cellular oxidative stress via perturbation of iron homeostasis and increased reactive oxygen species (ROS) production. In vivo validation studies such as tumor formation assays and treatment of established HNC xenograft models were also evaluated. The clinical relevance of UROD down-regulation in head and neck cancer was also demonstrated.

UROD knockdown has significant implications in the management of human cancers. Its over-expression is able to prognosticate for radiation resistance, thereby potentially allowing selection of cancer patients who would be suitable for siUROD radiosensitization. The therapeutic application of this approach is broad, and effective in the selective enhancement of radiation-induced cytotoxicity in cancer tissues, with no toxicity observed in normal tissues. Furthermore, there is a naturally occurring state of porphyria cutanea tarda (PCT), which is non-lethal; hence a "temporary" state of PCT would have minimal consequences to cancer patients during the few weeks of RT and/or chemotherapy. This discovery uncovers the translational significance of iron homeostasis and dysregulation within the context of tumor radiosensitization, warranting further investigations into this important biological process.

Therefore, in accordance with one aspect, there is provided a method for sensitizing a subject with cancer to a cancer therapy comprising administering to the subject a sensitizing amount of an agent that downregulates or inhibits UROD. Preferably, the cancer is a head and neck cancer and is selected from the group consisting of cancers originating from the lip, nasal and oral cavities, sinuses, pharynx, larynx, and other sites in this anatomical region.

In an embodiment, the cancer is selected from the group consisting of hypopharyngeal carcinoma, nasopharyngeal carcinoma, laryngeal carcinoma, lung adenocarcinoma, cervical carcinoma, prostate carcinoma and mammary adenocarcinoma.

In accordance with a further aspect, there is provided a method for sensitizing a subject with cancer to a cancer therapy comprising downregulating or inhibiting UROD in cancer cells of the subject.

In accordance with a further aspect, there is provided use of an agent that downregulates or inhibits UROD for sensitizing a subject to a cancer therapy.

In accordance with a further aspect, there is provided use of an agent that downregulates or inhibits UROD in the preparation of a medicament for sensitizing a subject to a cancer therapy.

In accordance with a further aspect, there is provided a compound for sensitizing a subject with cancer to a cancer therapy comprising a UROD inhibitor or UROD downregulator.

In accordance with a further aspect, there is provided a method for identifying an agent that sensitizes a subject with cancer to a cancer therapy comprising screening for a compound that downregulates or inhibits UROD.

In accordance with a further aspect, there is provided a method of prognosticating a survival outcome to a cancer therapy of a subject with cancer comprising: providing a sample comprising cancer cells from the subject; and determining the level of UROD expression and/or activity in the cancer cells; wherein a relatively low level of UROD expression and/or activity compared to a control is correlated with an improved clinical outcome in response to cancer therapy.

In accordance with a further aspect, there is provided a method of diagnosing a subject with cancer comprising: providing a sample from the subject; and assaying the level of UROD expression and/or activity in the sample; wherein a relatively high level of UROD expression and/or activity compared to a control is correlated with cancer.

In accordance with a further aspect, there is provided a kit for diagnosing a cancer in or prognosticating a survival outcome to a cancer therapy of a subject with the cancer, comprising an assay for UROD expression and/or activity along with instructions for use.

In accordance with a further aspect, there is provided a method for sensitizing a subject with cancer to a cancer therapy comprising elevating the intracellular iron in cancer cells of the subject.

In accordance with a further aspect, there is provided a method for sensitizing a subject with cancer to a cancer therapy comprising administering an agent that elevates intracellular iron.

In accordance with a further aspect, there is provided a use of an agent that elevates the intracellular iron in cancer cells for sensitizing a subject to a cancer therapy.

In accordance with a further aspect, there is provided a use of an agent that elevates the intracellular iron in cancer cells in the preparation of a medicament for sensitizing a subject to a cancer therapy.

In accordance with a further aspect, there is provided a compound for sensitizing a subject with cancer to a cancer therapy comprising an elevator of intracellular iron.

In preferable embodiments, the cancer therapy is radiation therapy. In one embodiment, the radiation therapy is therapy using ionizing radiation. In another embodiment, the radiation therapy is therapy using non-ionizing radiation and is preferably photodynamic therapy.

In other embodiments, the cancer therapy is chemotherapy. Preferably, the chemotherapy uses Cisplatin, 5-FU or Paclitaxel.

In some embodiments, the agent is any one of an siRNA, antisense oligonucleotide, miRNA, aptamer, protein, shRNA and small molecule, that downregulates or inhibits UROD or a modified version of any of the foregoing.

The term "radiation therapy" is used interchangeably with the term "radiotherapy". In some embodiments, the radiation is one of x-ray and gamma ray. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements may also be administered to expose tissues to radiation. However, any radiation therapy protocol can be used depending upon the type of cancer to be treated. Radiation therapy as used herein includes both ionizing and non-ionizing radiation. Non-ionizing radiation may be used, for example, in connection with photodynamic therapy ("PDT") and PDT-photosensitizing agents.

The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. Some examples of chemotherapeutic agents include, but are not limited to, antibiotic chemotherapeutics such as, Doxorubicin, Daunorubicin, Mitomycin (also known as mutamycin and/or mitomycin-C), Actinomycin D (Dactinomycin), Bleomycin, Plicomycin; plant alkaloids such as Taxol, Vincristine, Vinblastine; miscellaneous agents such as Cisplatin, VP16, Tumor Necrosis Factor; alkylating agents such as, Carmustine, Melphalan (also known as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard), Cyclophosphamide, Chlorambucil, Busulfan (also known as myleran), Lomustine; and other agents for example, Cisplatin (CDDP), Carboplatin, Procarbazine, Mechlorethamine, Camptothecin, Ifosfamide, Nitrosurea, Etoposide (VP16), Tamoxifen, Raloxifene, Estrogen Receptor Binding Agents, Gemcitabine, Navelbine, Farnesyl-protein transferase inhibitors, Transplatinum, 5-Fluorouracil, and Methotrexate, Temazolomide (an aqueous form of DTIC), or any analog or derivative variant of the foregoing.

As used herein, "UROD" refers to Uroporphyrinogen decarboxylase enzyme or gene as the context dictates. UROD is an enzyme in the heme biosynthetic pathway, catalyzing the decarboxylation of uroporphyrinogen to form coproporphyrinogen and four molecules of carbon dioxide.

The term "oligonucleotide" as used herein refers to a nucleic acid molecule comprising from about 1 to about 100 nucleotides, more preferably from 1 to 80 nucleotides, and even more preferably from about 4 to about 35 nucleotides. This may include nucleic acid molecules of variable length that correspond either to the sense strand or to the non-coding strand of a target nucleic acid sequence.

"Antisense oligonucleotides" (AON) are complementary to a region of a target gene and are capable of hybridizing to the target gene sequence and inhibiting gene expression. Gene expression is inhibited through hybridization of an AON to a specific messenger RNA (mRNA) sense target according to the Watson-Crick base pairing, typically in which adenosine and thymidine (uracil in mRNA) or guanosine and cytidine interact through hydrogen bonding. Without being bound to any theory, two mechanisms are generally thought to account for these effects, the first being hybridization with impaired translation of targeted mRNA, the second being the induction of RNase H or similar enzymes with associated degradation of target mRNA. Oligonucleotide compounds in accordance with the present invention also include siRNAs (small interfering RNAs) and the RISCs (RNA-induced silencing complexes) containing them that result from the RNAi (RNA interference) approach. The RNAi approach is a tool for the inhibition of target gene expression. RNAi is based on an ancient anti-viral defense mechanism in lower eukaryotes. It is induced by double-stranded RNA and its processing to typically 21-23 nt siRNAs, which cause the degradation of homologous endogenous mRNA after hybridizing to the target mRNA in a single stranded fashion with the assistance of the RISC complex. The way in which RNAi inhibits target gene expression remains to be fully elucidated, but presently, RNAi serves as an attractive choice approach to generate loss-of-function phenotypes across a broad spectrum of eukaryotic species, such as nematodes, flies, plants, fungi and mammals.

Oligonucleotide compounds in accordance with the present invention also include microRNA (miRNA). "MicroRNA" are single-stranded RNA molecules, typically of about 21-23 nucleotides in length, which regulate gene expression in a hybridization dependent manner. Typically, miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, typically at the 3' end of the mRNA, and their main function is to downregulate gene expression.

As used herein, the term "aptamer," e.g., RNA aptamer or DNA aptamer, includes single-stranded oligonucleotides that bind specifically to a target molecule. Aptamers are selected, for example, by employing an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment. Aptamers bind tightly and specifically to target molecules; most aptamers to proteins bind with a K.sub.d (equilibrium dissociation constant) in the range of 1 pM to 1 nM. Aptamers and methods of preparing them are described in, for example, E. N. Brody et al.

Mol. Diagn. 4:381-388.

In one embodiment, the subject aptamers can be generated using SELEX, a method for generating very high affinity receptors that are composed of nucleic acids instead of proteins. See, for example, Brody et al.

Mol. Diagn. 4:381-388. SELEX offers a completely in vitro combinatorial chemistry alternative to traditional protein-based antibody technology. Similar to phage display, SELEX is advantageous in terms of obviating animal hosts, reducing production time and labor, and simplifying purification involved in generating specific binding agents to a particular target PET.

An "amino acid" is a monomer unit of a peptide, polypeptide, or protein. There are twenty amino acids found in naturally occurring peptides, polypeptides and proteins, all of which are L-isomers. The term also includes analogs of the amino acids and D-isomers of the protein amino acids and their analogs.

A "protein" is any polymer consisting essentially of any of the 20 amino acids. Although "polypeptide" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and is varied. The term "protein" as used herein refers to peptides, proteins and polypeptides, unless otherwise noted.

The term "small molecule" is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic or inorganic molecules, and natural product extract libraries.

The term "downregulate" is used herein to refer to at least partial inhibition or knockdown of the expression of a gene or activity of the protein that it encodes. For example, in some embodiments, an antisense oligonucleotide, siRNA or miRNA compound exhibiting complementarity to UROD downregulates or inhibits expression of UROD in a hybridization dependent manner. In another embodiment an aptamer, protein or small molecule downregulates or inhibits UROD protein activity by binding thereto.

As used herein, the term "screening" or "to screen" refers to a process in which a large number of potentially useful agents are processed in the methods of the invention. Without limitation, screening may refer to an assay of members having a desired activity or function from a library such as small molecule, aptamer, protein and nucleic acid libraries. For example, in some embodiments, potential antisense oligonucleotides, siRNAs and/or miRNAs exhibiting complementarity to UROD are screened/processed in order to identify species that downregulate or inhibit expression of UROD in a hybridization dependent manner. In another embodiment aptamers, proteins and/or small molecules are screened/processed to identify species that downregulate or inhibit UROD protein activity by binding thereto.

The term "sensitizing amount" means a sufficient amount of an agent to provide the desired sensitizing effect. For example, in some embodiments, "sensitizing amount" means that dose of agent effective to increase the sensitivity of cancerous cells or tumour to radiation therapy or chemotherapy.

The term "prognosticating" as used herein means predicting or identifying the clinical outcome group that a subject belongs to according to the subject's similarity to a control group or control profile.

The term "diagnosing" means judging, predicting, assessing and/or evaluating as well as identifying and characterizing, including screening, whether a person is susceptible of or suffers from cancer, including, but not limited to head and neck cancers.

The term "sample" as used herein refers to any fluid, cell or tissue sample from a subject, which can be assayed, for example, for UROD expression or activity.

As used herein, the term "control" refers to a specific value or dataset that can be used to prognosticate, diagnose or classify the value e.g. expression level of UROD obtained from the test sample associated with an outcome class (e.g. high vs. low survival or tumour vs. normal cells). A person skilled in the art will appreciate that the comparison between the expression of UROD in the test sample and the expression of UROD in the control will depend on the control used. In some embodiments, the control comprises an UROD expression profile from multiple samples in order to dichotomize the control values into different outcome classes (e.g. high vs. low survival or tumour vs. normal cells). As such, when a test sample is compared to the UROD expression profile, the test sample can be placed in one of the outcome classes based on UROD expression.

Examples

Materials and Methods

Cell Lines

FaDu, A549, SiHa, ME-180, T47D, DU-145, and MRC5 cells were obtained from the American Type Culture Collection (Manassas, Va.). Normal human oropharyngeal (NOP) and oral epithelial (NOE) cells were purchased from Celprogen (San Pedro, Calif.). Untransformed fibroblasts from familial porphyria cutanea tarda (type II) patients (GM01482, GM00977, GM00961, GM01041) and GM05757 (primary normal human skin) fibroblasts were obtained from Coriell Institute (Camden, N.J.). All cell lines were cultured according to the manufacturer's specifications. C666-1 undifferentiated nasopharyngeal cancer cells [16] were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Wisent, Quebec, Canada) and antibiotics (100 mg/L penicillin and 100 mg/L streptomycin). UTSCC-8 and -42a laryngeal squamous cell cancer cells were a gift from R. Grenman (Turku, Finland) and maintained as previously described [17]. All cells were maintained in 5% CO.sub.2, 21% O.sub.2, and 95% humidity at 37.degree. C. unless otherwise stated.

Patient Samples

Thirty-eight formalin-fixed paraffin-embedded (FFPE) tissue biopsies from locally advanced HNSCC patients (Stage III or IV; oropharynx, hypopharynx, or larynx primary SCC subsites), who participated in a randomized clinical study of two RT fractionation regimens [18] were utilized with Institutional Research Ethics Board approval. FFPE samples were macro-dissected for regions of invasive SCC (>70% malignant epithelial cell content). Five normal human larynx and tonsillar FFPE tissues were purchased from Asterand (Detroit, Mich.). Total tumor RNA was extracted with RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion, Austin, Tex.) as specified by the manufacturer.

Reagents

Cisplatin, 5-fluorouracil, Paclitaxel, .delta.-aminolevulinic acid hydrochloride, and deferoxamine mesylate salt were obtained from Sigma-Aldrich (St. Louis, Mo.). All compounds were dissolved and/or diluted in complete media.

BrdU-Based siRNA High-Throughput Screen

The Human siGENOME Druggable and Protein Kinase siRNA Libraries (Dharmacon, Lafayette, Colo.) were provided by the Samuel Lunenfeld Research Institute (SLRI) HTS Robotics Facility (Toronto, Canada). Automation of the 96-well siRNA transfection and bromodeoxyuridine (BrdU) cell proliferation assay (Exalpha Biologicals, Shirley, Mass.) were performed using the BioMek FX (Beckman Coulter, Fullerton, Calif.), SpectraMax Plus.sup.384 microplate reader (Molecular Devices, Sunnyvale, Calif.), and SLRI robotics platform.

Working stock solutions of siRNA were prepared in Opti-MEM I reduced-serum media (Invitrogen, Carlsbad, Calif.). Reverse transfections (final concentration of 40 nM siRNA) were performed with Lipofectamine 2000 (Invitrogen) as specified by the manufacturer. Columns 1 and 2 of each plate contained siRNA targeting DNA ligase IV (LIG4 siGENOME SMARTpool; Dharmacon), serving as the positive radiosensitizing control, and scrambled negative siRNA control (ON-TARGETplus Non-Targeting Pool; Dharmacon), respectively. Twenty-four h post-transfection, 100 .mu.L of complete media was added to each well, then cells were irradiated using a .sup.137Cs unit (Gammacell 40 Extractor; MDS Nordion, Ottawa, Canada) at a dose rate of 0.84 Gy/min. Cells were incubated for an additional 72 h, at which time, BrdU (Exalpha Biologicals) was added to each well. After 24 h, cells were monitored for BrdU incorporation on a SpectraMax Plus.sup.384 microplate reader according to the manufacturer's specifications.

Transfections

siRNAs targeting UROD (Hs_UROD.sub.--2/8 HP GenomeWide siRNAs) and a scrambled control (AllStars Negative Control siRNA) were purchased from Qiagen (Valencia, Calif.). A plasmid vector containing the protein-coding sequence of UROD (Hs_UROD_IM.sub.--1 QIAgene Expression Construct) and an empty vector control (pQE-TriSystem Vector) were also purchased from Qiagen. All transfections were performed in complete media without antibiotics using Lipofectamine 2000 and 40 nM of siRNA and/or 1 .mu.g of plasmid DNA.

The description continues in the full USPTO document.